CN104390946A - Method for determining content of ochratoxin A in juice - Google Patents
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Abstract
The invention discloses a method for determining the content of ochratoxin A in juice. The method comprises the following steps: pretreating a sample by adopting a simple liquid-liquid extraction and purification step; scanning with parameters including optimized scanning wavelengths, scanning intervals and the like, and acquiring three-dimensional fluorescent data of a standard product and the sample; carrying out mathematic separation treatment on the obtained data by adopting a parallel factor analyzing method (PARAFAC); establishing a correction model by using the standard product with the known concentration and by combining mathematic separation with chemical and physical separation; and predicating a component to be detected under the conditions that unknown and uncorrected background interferences are included and spectrums are seriously overlapped. The method is simple and rapid, and has the high sensitivity; and the content of ochratoxin in the juice can be determined under the unknown background interferences. The method belongs to the field of food safety.
Description
Technical field
The present invention relates to a kind of method of ochratoxin A content, be specifically related to the method measuring ochratoxin A content in fruit juice based on three-dimensional fluorescence second order correction method, belong to field of food safety.
Technical background
Ochratoxin A (OTA) is a kind of by mycetogenetic toxic metabolic products, is common in Grain and its product, coffee, and in fruit and goods thereof, international cancer research institution (IARC) has been defined as 2B class carcinogenic substance.China does not also have the clear and definite limit standard of ochratoxin A in fruit juice at present, and especially its rapid analysis and test method research is less.
The main detection method of ochratoxin A has thin-layered chromatography, euzymelinked immunosorbent assay (ELISA), immune affinity column chromatography to purify fluorimetry and SPE HPLC, liquid-liquid extraction purification high performance liquid chromatography etc.In ochratoxin A structure, there is conjugated double bond, fluorescence can be sent under UV-irradiation, so detection method comparatively conventional is at present high performance liquid chromatography fluorescence detector method, but when multi-component complex matrix chemical system is analyzed, first must carry out physics or Chemical Decomposition to system sample components, and then utilize the retention time difference of chromatographic column to analyze to target analytes, it is loaded down with trivial details that this disposal route can allow whole analytic process become usually, time cycle is longer, and the low cost be not suitable in enterprise's production, the requirement that a large amount of sample detects fast.
Fluorescence spectrophotometry instrument relative cost is lower, has simple to operate, and detection speed is fast, sensitivity advantages of higher.But what measure is total emitting fluorescence value, poor selectivity is measured to one matter in complex matrices, because the fluorescence spectrum of ochratoxin A is overlapping with the spectral mixing of complex sample matrix in fruit juice, thus without being separated direct the method, to detect the feasibility of wherein ochratoxin A content poor, and namely conventional fluorescent analytic approach is difficult to meet analysis requirement.
Three-dimensional data information is obtained through fluorescence spectrometry by after simple for sample pre-treatment purifying, in conjunction with chemometrics method with " mathematics separation " combination " Chemical Decomposition " to improve its selectivity, can when containing unknown, non-correcting background interference and spectrum overlapping, realize the direct quantitative determination to component to be measured, in fields such as Safety of Food Qualities, there is important analytic potential.
Summary of the invention
The present invention discloses a kind of method measuring ochratoxin A content in fruit juice, based on three-dimensional fluorescence second order correction method, be separated the method carrying out the ochratoxin A content in Fast Measurement fruit juice in conjunction with Chemical Decomposition with mathematics, aim to provide a kind of method of low cost, Fast Measurement.
The present invention adopts following technical scheme:
The present invention adopt excite-emitting fluorescence matrix (EEM) combines with parallel factor method (PARAFAC), after sample simple process, ochratoxin A content in analysis fruit juice product.
Measure a method for ochratoxin A content in fruit juice, comprise the steps:
Step (1) Modling model: based on parallel factor method (PARAFAC), preparation ochratoxin A corrects sample to set up the calibration model of quantitative test;
At correction sample, checking sample, the fruit juice extract of linear latitude of formulation ochratoxin A, and gather its three-dimensional fluorescence excitation-emission matrix spectrum data under scanning wavelength after optimization and sweep spacing, to avoid the interference of Rayleigh scattering, Raman scattering, reduce the interference of redundancy SPECTRAL REGION and the very low SPECTRAL REGION of signal to noise ratio (S/N ratio); Adopt parallel factor method to carry out mathematics to obtained three-dimensional data battle array and be separated parsing, and set up calibration model;
Step (2) verification model: with ochratoxin A checking sample, positive model for school building is judged, and verify the reliability of calibration model;
Prepare with the disposal route identical with correcting sample and verify sample, and gather three-dimensional fluorescence data through fluorescent scanning, analyze through parallel factor method, after calibration model prediction, obtain prediction concentrations, and with theoretical concentration comparative analysis with the reliability of judgment models;
Step (3) analysis measures: measure the content express-analysis of ochratoxin A in fruit juice actual sample with calibration model;
With the fruit juice of different cultivars for actual sample, fruit juice actual sample, after liquid-liquid extraction pre-treatment, gathers three-dimensional fluorescence spectrum data matrix under identical experiment parameter, obtains the content of ochratoxin A in actual sample through mathematics separation and calibration model prediction.
On technique scheme basis, further,
In described step (1), ochratoxin A corrects the sample range of linearity and is: 0.27 ~ 3.24ng/mL;
In described step (1), the scanning wavelength optimized and sweep spacing: excitation wavelength range is 285 ~ 360nm, sweep spacing 5nm; Emission wavelength ranges is 420 ~ 510nm, sweep spacing 5nm;
In described step (2), to the concentration range of the ochratoxin A checking sample solution preparation that model is verified be: 0.44 ~ 2.18ng/mL;
In described step (3), the spiked levels of fruit juice actual sample is: 0 ~ 8.89ng/mL, and the concentration after ensureing to extract is within the range of linearity;
In described step (3), liquid-liquid extraction pre-treatment step is: fruit juice actual sample is through methylene chloride liquid-liquid extraction, and centrifugal layering, gets methylene chloride through dilute sodium bicarbonate solution back extraction, after centrifugal layering, get upper strata aqueous phase, add hcl acidifying, bubble removing is also preserved to be measured.Blank assay is not for add target samples of juice.
Measure the performance evaluation of ochratoxin A content method in fruit juice:
After mathematics is separated, relative excitation spectrum, relative emission spectra and background interference spectrum are obtained to fruit juice actual sample, and compares analysis with the relative excitation spectrum of ochratoxin A standard items, emission spectrum, see its similarity degree; To mark-on fruit juice after pre-treatment is separated with mathematics, obtain prediction concentrations and the recovery, with sensitivity (SEN), selectivity (SEL), Monitoring lower-cut (LOD), the quality factors (FOM) such as prediction residual root mean square (RMSE), with verification method, the accuracy of appraisal procedure.
The basis of chemometrics application, three linear components models:
Suppose that the standard specimen of mensuration and the total number of samples of pre-test sample are K, excitation wave long number is I, and transmitted wave long number is J.For the 3D fluorescence response number gust X (I × J × K) that 1 collects, element (i, j, k) wherein represent sample k excitation spectrum number be i, emission spectrum number be j time fluorescence intensity, it meets three linear components models below:
wherein: i=1,2 ..., I; J=1,2 ..., J; K=1,2 ..., K
N represents has the number of components of actual contribution (comprising object, total number of components of background and mutual interference) to fluorescence response; X
ijkthe element (i, j, k) in 3D fluorescence response number gust X, it represent sample k excitation spectrum number be i, emission spectrum number be j time fluorescence intensity; C
knit is the element (k, n) in relative concentration battle array C (K × N); a
init is the element (i, n) in relative excitation spectrum battle array A (I × N); b
jnthe element (j, n) in relative emission spectra battle array B (J × N), e
ijkit is the element (i, j, k) in 3D residual error number gust E (I × J × K).
Can find out that three-dimensional data battle array X has the uniqueness of three linear decomposition from above-mentioned formula, under unknown disturbances exists, relative excitation matrix A, relative transmission matrix B and relative concentration Matrix C in K sample can be obtained.Second order correction method has unique " second order advantage ", can realize unknown disturbances component coexist lower to complicated multicomponent analysis system in the advantage of fast quantitative analysis of target components, this feature makes three linear components models can be applicable to actual sample analysis.
The determination of number of components:
Number of components in three-dimensional data refers to the smallest group mark of the parsing of matching exactly needed for trilinear model, has both comprised component to be measured, and has also comprised the interfering component coexisted with it.Corcondia is used to the method determining component, the method estimates number of components by the similarity degree calculated in parallel factor analysis model between superdiagonal matrix T and least square fitting battle array G, the method is called as core consistent diagnosis determination number of components, and formula is as follows:
Wherein, F is the number of components of model; g
deffor the element of three-dimensional matrice G (least square fitting battle array); t
deffor the element of three-dimensional matrice T (super diagonal matrix).For desirable PARAFAC model (it is suitable that number of components is selected), super diagonal matrix and least square fitting battle array should be closely similar, and core homogeneity value now will equal 100%.Usually, think that model is linear close to three when core homogeneity value is more than or equal to 60%.But when core homogeneity value is less than 60%, then think depart from three linear.So, can according to the number of components of the situation of change judgement sample of core homogeneity value.
The analysis of quality factor:
The quality analyzing this experiment mainly contains the accuracy that sensitivity (SEN), selectivity (SEL), Monitoring lower-cut (LOD) and prediction residual root mean square (RMSEpred) check second order correction method to predict the outcome.
In second order correction, the estimation analyzing quality factor is closely related with the calculating of pure analytic signal.Sensitivity refers to the pure analytic signal of unit concentration, and selectivity refers to the ratio of sensitivity and resultant signal, calculates herein according to following formula:
SEL＝{[(A
TA)-1]
nn*[(B
TB)
-1]
nn}
-1/2
SEL＝K{[(A
TA)
-1]
nn*[(B
TB)
-1]
nn}
-1/2
Wherein: subscript n n is the individual element of matrix (n, n); K is the resultant signal of component n when unit concentration (concentration scoring parameters)
LOD＝3.3s(0)
Wherein: S (0) is the prediction concentrations standard deviation of three background blank samples
Wherein: k is sample number, C
actfor actual concentrations, C
predfor prediction concentrations
If RMSE is less, predicted value is more close to theoretical value, then precision of prediction is higher, and RMSE can be utilized to assess the predictive ability of calibration model.
In described performance evaluation: sensitivity (SEN) refers to the pure analytic signal of unit concentration, selectivity (SEL) refers to the ratio of sensitivity and resultant signal.
Beneficial effect of the present invention is:
By simple liquid-liquid extraction step, pre-service is carried out to sample, then scan under the parameter such as scanning wavelength and sweep spacing after optimization and gather the three-dimensional fluorescence data of standard items and sample, parallel transport (PARAFAC) is adopted to carry out mathematics separating treatment to the data of gained, chemistry and physical separation is combined with " mathematics separation ", the standard items of concentration known are utilized to set up calibration model, when containing unknown, non-correcting background interference and spectrum overlapping, realize the prediction to component to be measured.The method is simple, fast, highly sensitive, the mensuration of ochratoxin content in fruit juice under Unknown Background interference can be realized, and there is unknown disturbances coexist and also do not affect the advantage of measured portions analysis result to be measured.
Accompanying drawing explanation
When Fig. 1 background interference exists, the samples of juice resolution figure (N=2) obtained by PARAFAC method
A () exciting light is differentiated figure (b) utilizing emitted light and is differentiated figure
In figure: 1. real spectrum, 2. differentiate the ochratoxin A spectrum obtained, 3. background interference
Fig. 2 is the relative concentration resolution figure that PARAFAC algorithm (N=2) is differentiated
In figure: 1. ochratoxin A relative concentration; 2. background interference,
Sample 1-8 is for correcting sample, and sample 9-12 is actual sample
Embodiment
Below, in conjunction with specific embodiments, invention is further described.
Embodiment:
One, operation steps
Step (1), based on parallel factor method (PARAFAC), sets up the calibration model of quantitative test according to standard items
1), the preparation of standard items:
Get 1mg ochratoxin A standard items hplc grade methanol to dissolve completely, be settled to 50mL (20 μ g/mL ,-20 DEG C keep in Dark Place), as ochratoxin A standard reserving solution.
Get 0.5mL ochratoxin A standard reserving solution, by methanol constant volume to 100mL, concentration is that 100ng/mL (4 DEG C keep in Dark Place) is stand-by; The ochratoxin A solution of a series of variable concentrations is prepared with dilute sodium bicarbonate solution, scanned samples 3D fluorescence intensity one by one, carries out range of linearity investigation, and it is in 0.27 ~ 3.24ng/mL concentration range, related coefficient is 0.99, and linear relationship well can carry out quantitative test.
2) preparation of sample, is corrected: with dilute sodium bicarbonate solution preparation ochratoxin A working fluid, make ochratoxin A concentration range between 0.27 ~ 3.24ng/mL;
Utilize fluorospectrophotometer to correction sample and checking sample scanning collection data, for effectively avoiding the interference of Rayleigh scattering, Raman scattering, reduce the interference of redundancy SPECTRAL REGION and the low SPECTRAL REGION of signal to noise ratio (S/N ratio), selective excitation wavelength coverage is 285 ~ 360nm, emission wavelength ranges is 420 ~ 510nm, interval 5nm image data respectively, slit width is 5.0/5.0nm, and sweep velocity is 12000nm/min.Under the parameter of setting, the three-dimensional fluorescence spectrum data of acquisition correction sample and checking sample, build three-dimensional data battle array to be analyzed.
Utilize the consistent diagnosis of core (corcondia) to carry out rand estination to three obtained dimension battle arrays (19 × 16 × 14), when number of components≤2, core homogeneity value is greater than 60%; And as number of components >2, core homogeneity value reduces.This illustrates in this system, and when number of components is 2, model is linear closest to three.Therefore, when predicting checking sample, selected number of components is 2.
The preparation of step (2) checking sample: preparation checking sample (do not contain and disturb) is used for investigating the stability of calibration model, the concentration range of ochratoxin A checking sample is included in and corrects within sample concentration range.
Parallel factor method (PARAFAC) is adopted to resolve three-dimensional matrice, linear regression is carried out to concentration, when analyzing the checking sample of spiked levels 0.44 ~ 2.18ng/mL, this arithmetic analysis is verified the prediction concentrations of sample and adds concentration closely really.The average recovery rate of ochratoxin A is 97.38% ~ 103.14%, RSD<8%, RMSEpred=0.0241ng/mL, and for the checking sample of preparation, PARAFAC gives satisfied result, illustrates that set up model is reliable.
Table 1 PARAFAC method measures ochratoxin A result in checking sample
Step (3), measures the express-analysis of ochratoxin A content in fruit juice actual sample
By samples of juice, get a certain amount of mark-on fruit juice actual sample through methylene chloride liquid-liquid extraction, centrifugal layering, removing upper strata aqueous phase, gets the methylene chloride of certain volume through dilute sodium bicarbonate solution back extraction, after centrifugal layering, get upper strata aqueous phase, add hcl acidifying, bubble removing is also preserved to be measured.Blank assay is the sample not adding ochratoxin A standard items.To the multiple scanning three times under the instrument condition determined of the sample after process, and gather its three-dimensional fluorescence spectrum data matrix.
Adopt core unanimously to analyze to sample, estimate that system number of components is 2, one of them component contributed by object ochratoxin A, and another component contributed by the background interference of matching.Through mathematical analysis, obtain relative excitation spectrum battle array, relative emission spectra battle array and relative concentration battle array, obtain the content of ochratoxin A in fruit juice.It predicts the outcome, and (representing with the recovery) is as shown in table 2:
Table 2 PARAFAC method measures the result of ochratoxin A in samples of juice
Two, the performance evaluation of method
After PARAFAC mathematics is separated, relative excitation spectrum is obtained to mark-on actual sample, relative emission spectra, with background interference spectrum, and compare analysis with the relative excitation of ochratoxin A standard items, emission spectrum, see its similarity degree, as shown in Figure 1, when number of components is 2, it is similar to real spectrum respectively with emission spectrum that PARAFAC algorithm differentiates the ochratoxin A excitation spectrum obtained, illustrate that the solution that these algorithm models obtain is reliable, the ochratoxin A contained in fruit juice can be differentiated, also embody the uniqueness of three linear decomposition simultaneously.Also can find out, what ochratoxin A was maximum excites, emission wavelength greatly about 330,460nm place, match with ochratoxin A standard items fluorescent scanning result.
From relative concentration Fig. 2, correct impurity content in sample and be almost 0, in actual sample, impurity content is higher, and fluorescence interference and the fluorescence overlapping of target analytes of endogenous substance, have a great impact the quantitative measurement of object.Need the fast quantitative analysis that the means be separated through mathematics could realize object.
With sensitivity (SEN), selectivity (SEL), Monitoring lower-cut (LOD), the quality factors (FOM) such as prediction residual root mean square (RMSE), with the accuracy of verification method.
Table 3PARAFAC method measures samples of juice measurement result quality factor and analyzes
| Quality factor | Fruit juice |
| SEL | 0.552 |
| SEN(mL/ng) | 0.104 |
| LOD | 0.081 |
| RMSE(ng/mL) | 0.164 |
The foregoing is only better possible embodiments of the present invention, not thereby limit to the scope of the claims of the present invention, therefore the equivalence change that every utilization content of the present invention is done, be all contained in protection scope of the present invention.
Claims (9)
1. measure a method for ochratoxin A content in fruit juice, comprise the steps:
Step (1) Modling model: based on parallel factor method (PARAFAC), preparation ochratoxin A corrects sample to set up the calibration model of quantitative test;
Step (2) verification model: with ochratoxin A checking sample, positive model for school building is judged, and verify the reliability of calibration model;
Step (3) analysis measures: measure the content analysis of ochratoxin A in fruit juice actual sample with calibration model.
2., according to the method measuring ochratoxin A content in fruit juice described in claim 1, it is characterized in that,
Described step (1), at correction sample, the checking sample of linear latitude of formulation ochratoxin A, takes fruit juice extract, and under the scanning wavelength optimized and sweep spacing, gathers its three-dimensional fluorescence excitation-emission matrix spectrum data; Adopt parallel factor method (PARAFAC) to carry out mathematics to obtained three-dimensional data battle array and be separated parsing, and set up calibration model.
3., according to the method measuring ochratoxin A content in fruit juice described in claim 1, it is characterized in that,
Described step (2), with the disposal route preparation checking sample identical with correcting sample, and gathering three-dimensional fluorescence data through fluorescent scanning, analyzing, obtain prediction concentrations through parallel factor method, and the reliability of judgment models.
4., according to the method measuring ochratoxin A content in fruit juice described in claim 1, it is characterized in that,
Described step (3), fruit juice actual sample is after liquid-liquid extraction pre-treatment, under identical experiment parameter, gather three-dimensional fluorescence spectrum data matrix, obtain the content of ochratoxin A in actual samples of juice through the analysis of parallel factor method and calibration model prediction.
5. according to the method measuring ochratoxin A content in fruit juice described in claim 1, it is characterized in that, in described step (1), ochratoxin A corrects the sample range of linearity and is: 0.27 ~ 3.24ng/mL.
6. according to the method measuring ochratoxin A content in fruit juice described in claim 2, it is characterized in that, the scanning wavelength of optimization and sweep spacing: excitation wavelength range is 285 ~ 360nm, sweep spacing 5nm; Emission wavelength ranges is 420 ~ 510nm, sweep spacing 5nm.
7. according to the method measuring ochratoxin A content in fruit juice described in claim 3, it is characterized in that, to the concentration range of the ochratoxin A checking sample that model is verified be: 0.44 ~ 2.18ng/mL.
8. according to the method measuring ochratoxin A content in fruit juice described in claim 4, it is characterized in that, the spiked levels of fruit juice actual sample is: 0 ~ 8.89ng/mL, and the concentration after ensureing to extract is within the range of linearity.
9. according to the method measuring ochratoxin A content in fruit juice described in claim 4, it is characterized in that, liquid-liquid extraction pre-treatment step is: samples of juice is through methylene chloride liquid-liquid extraction, centrifugal layering, get methylene chloride through dilute sodium bicarbonate solution back extraction, after centrifugal layering, get upper strata aqueous phase, add hcl acidifying, bubble removing is also preserved to be measured.
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